Infective endocarditis (IE) is a serious cause of morbidity and mortality in the US. This condition is caused by infection with bacteria or fungi that invade the bloodstream and adhere to the heart, causing infection. The use of appropriate antibiotics is crucial for eradicating the infection. Typically blood culture is used to determine the presence and identify of the microbe responsible. However, in some cases cultures are negative, due to prior treatment with antibiotics, or to the involvement of fastidious organisms (many of which are especially associated with infective endocarditis). Culture-negative cases represent a very serious problem, since they are difficult to conclusively diagnose in the absence of demonstrated infection, and no information is available to guide the physician in making antibiotic choices. A large fraction of patients suffering from IE eventually need surgery to replace damaged heart valves, and even when infected tissue is available for testing, microbial ID may be difficult to determine by conventional culture-based methods. The aim of the proposed project is to develop a molecular test for detection and identification of microorganisms causing infective endocarditis. The diagnostic test will be based on a reverse line blot assay developed by investigators, which enables detection and identification of a wide range of common bacterial pathogens. In contrast with the 2-3 day delay required for culture-based methods, it will offer same-day turnaround by obtaining genetic information directly from clinical samples without need for bacterial growth. Thus it will allow detection and identification of the organism in culture-negative as well as culture-positive cases. In Phase I, the assay will be developed based on blood and tissue samples from patients with suspected bacterial or fungal bloodstream infections, including IE. Results of the reverse line blot method will be compared with those of conventional culture-based ID. We will also develop an extension of the technology for detection of some of the antibiotic resistance genes most relevant to IE (MRSA and VRE). In Phase II, a commercial version of the assay will be completed and its accuracy and sensitivity in providing microbial ID, and thereby potentially improving treatment decisions and healthcare outcomes for affected patients will be evaluated in a prospective study. Overall, this novel molecular diagnostic assay will provide a powerful tool in efforts to improve diagnosis and treatment of infective endocarditis. PUBLIC HEALTH RELEVANCE: Infective endocarditis is an important cause of morbidity and mortality in the US. Culture-negative cases, resulting from prior antibiotic use or fastidious microorganisms, are especially difficult to diagnose and treat. This project will result in the development of a rapid test for simultaneous detection and identification of bacteria and fungi responsible for infective endocarditis, and will be effective even for culture-negative cases. This will enable clinicians to accurately diagnose the infection and select more effective antibiotic therapy, improving the survival rate for affected patients.